The overall goal of this project is to understand the in vivo function of histones and their role in chromatin differentiation. These studies are greatly facilitated by the remarkable nuclear dimorphism of Tetrahymena in which transcription occurs specifically in vegetative somatic macronuclei and mitosis and meiosis occurs only in germline micronuclei, We are performing in vivo analyses of histone function using methods we developed for mass transformation, which occurs by homologous integration, facilitating gene knockout and gone replacement in the germline micronucleus, the somatic macronucleus, or both. T. thermophila may be second only to Saccharomyces cerevisiae in the depth to which its histone primary sequence variants and secondary modification sites have been characterized and the degree to which these have been associated with nuclear processes in different physiological and developmental states. Importantly, some of the features of the Tetrahymena histone complement and its modifications resemble those of mammals that are lacking in S. cerevisiae. Assisted by the newly available sequence of the Tetrahymena (macronuclear) genome, we have identified all of the genes that encode histones. These include three new genes whose function we propose to study: novel, highly divergent H2A, a likely H3 replacement variant and a likely centromereic H3 homologous to Cenp-A. We will continue studies on the function of phosphorylation of linker histones, proposing to test our hypothesis that it regulates gone expression by site-specific de-phosphorylation in chromatin. We will test the hypothesis that H2A.Z has a general function to limit the spread of telomeric heterochromatin. We will analyze the functions of phosphorylation and ubiquitination of H2A and H2B, determining whether these modifications have redundant or unique functions. We will test the hypothesis that phosphorylation of specific residues flanking the H2A ubiquitinafion site serves as a binary switch to regulate ubiquitination. We will use our large array of Tetrahymena strains expressing site-specific mutations of histone modification sites, coupled with TAP tagging, to identify proteins that recognize these modifications. Finally, using mutations in genes encoding molecules that function in the RNAi machinery required for heterochromatin formation, we will test the hypothesis that the centromeric H3 is targeted to centromere DNA sequences by an RNAi-like mechanism. Given the conservation of histones and their secondary modifications, and their demonstrated importance in disease, we expect the studies proposed here to provide important insights into the functions of linker histones, histone variants and histone modification in transcription and chromosome function in eukaryotes. [unreadable] [unreadable]